KR20200011490A - Enhancement of brain function, food and medicine using lipopolysaccharide - Google Patents

Abstract

As a result of oral administration of Pantoea LPS to mice with Alzheimer's disease, the results of the investigation revealed that oral administration of Pantoea LPS significantly reduced Aβ peptide accumulation in the brain and improved learning function. Thereby, the brain function improving agent or the food composition which has the inhibitory effect of A (beta) intracranial deposition or the improvement effect of learning function which is easily available is provided.

Description

Enhancement of brain function, food and medicine using lipopolysaccharide

The present invention relates to brain function improving agents, foods and pharmaceuticals containing lipopolysaccharide.

In recent years, the total population has decreased in Japan, while the elderly population 65 years or older tend to increase year by year. According to the Ministry of Health, Labor and Welfare, the elderly population (2015) is over 33 million, and the aging rate reaches 26.7%. In dementia, one of the elderly diseases, the number of patients in Japan is currently over 4.6 million, and it is expected that by 2025, 7 million people will be 1 in 5 people. About 60% of dementia patients are Alzheimer's disease, about 20% are vascular dementia, and the remainder includes several dementia diseases such as Lewy body dementia. Clinical symptoms of Alzheimer's disease include cognitive impairments such as memory and speech impairments, mental symptoms, and behavioral disorders. As a pathological feature of Alzheimer's disease, abnormal structures such as amyloid plaques and neurofibrillary tangles, called amyloid plaques, are deposited in a wide range of cerebral cortex and hippocampus. The major component of amyloid plaques is amyloid β-protein (hereinafter also referred to simply as “Aβ”). In particular, Aβ peptides (hereinafter referred to simply as “Aβ1-42”) consisting of 42 residues are highly cohesive and are the core for amyloid deposition. It is known to play a role. The latter is excessively phosphorylated tau protein. Currently, the amyloid hypothesis is generally supported as a pathological cascade of Alzheimer's disease. Formation of amyloid plaques due to excessive accumulation of Αβ, accumulation of tau protein (neurofibrillary fibrous change), followed by neurodegeneration and neuronal cell death (Non-Patent Document 1). Aβ appears in the brain of a normal person, for example, but it is believed that Aβ accumulation is promoted by gene mutation of an enzyme involved in Aβ production or by deactivation of the Aβ degradation system. As drugs for treating Alzheimer's disease, drugs that target neurotransmitters and their receptors (acetylcholinesterase inhibitors and N-methyl-D-aspartic acid receptor antagonists) have been developed, but accumulation of Aβ and aggregation of tau protein Because it is not a cure for treating pathological changes in the back, it is not a fundamental cure that can arrest the progression of Alzheimer's disease.

On the other hand, microglia, which are macrophages of brain tissue, have been shown to play an important role in maintaining homeostasis in the brain, such as repairing damaged tissues and eliminating waste products from the brain. Since microglia emits Aβ receptor and ingests and degrades Aβ by a phagocytosis reaction, it is expected to prevent Alzheimer's disease due to excessive accumulation of Aβ by activating Ag phagocytic activation of microglia. Can be. Recently, the search for a compound having a function of activating the Aβ phagocytosis ability of microglia has been progressed, and food ingredients such as oleic acid amide (non-patent document 2) and peptide (patent document 1) are effective in in vitro experimental systems. Is being reported.

By the way, lipopolysaccharide is one of the outer membrane components of the cell wall of Gram-negative bacteria. The name endotoxin was given in 1892 for cholera-derived toxic substances, and it was reported in 1954 that the main body of the endotoxin was lipopolysaccharide (Non-Patent Document 3). Recently, however, intracranial injection of lipopolysaccharide into Alzheimer's disease model mice has been reported to induce microglia activation and inhibit the deposition of Aβ in the brain (Non-Patent Document 4). Similarly, intraperitoneal injection of lipid A, a constituent of lipopolysaccharide and applied as an adjuvant (immunostimulator), has been reported to show inhibition of Aβ brain deposition and improvement of learning function. (Nonpatent literature 5). On the other hand, ingestion by lipopolysaccharide orally or transdermally, no clear toxicity is confirmed, and there is a report showing improvement effect on diseases such as hyperlipidemia and allergy (Non Patent Literature 6). Soma et al. Discovered that one of these lipopolysaccharides is lipopolysaccharide (hereinafter also referred to as `` pantoea bacterium LPS '') derived from wheat symbiosis bacteria Pantoea agglomerans. The fermentation and culture method for obtaining the lipopolysaccharide which is safe even if added to food etc. was reported (patent document 2). Fukasaka et al. Reported in animal experiments that sublingually administered pantoea bacteria LPS acted as an adjuvant to increase mucosal immune activity (Non-Patent Document 7). In addition, Kobayashi et al. Reported that pantoea bacteria LPS improves the phagocytosis of Aβ1-42 by examination using primary cells of mouse brain-derived microglia (Non-Patent Document 8). However, orally administered lipopolysaccharide is hardly absorbed from the digestive tract. If this is, for example, starch, humans are digested because they have enzymes (amylase, maltase) that digest starch (the α1-4 -binding macromolecule of glucose) (Wikipedia, starch), but LPS polysaccharide is 3 The structure consists of five types of hexasaccharides and pentoses (Wikipedia, Lipopolysaccharides), and since humans do not have digestive enzymes capable of degrading the polysaccharide structure of LPS, they are not digested. In addition, regarding the transition to the brain, even if lipopolysaccharide is absorbed into the blood by oral administration, for example, the efficiency in the brain also decreases, so the lipopolysaccharide administered orally does not reach the brain. do. Needless to say, the effect of inhibiting A [beta] deposition in the brain or the improvement of learning function by oral administration of lipopolysaccharide has not been reported so far and is not described in any literature.

Japanese Patent Application Laid-Open No. 2016-193865 Japanese Patent Application Laid-Open No. 2011-193877

J. Hardy et al., Science, 297, 353-356, 2002 Y. Ano et al., PLOS ONE, 10, e0118512, 2015 O. Westpal et al., Angewandte Chemie, 66, 407-417, 1954 D. L. Herber et al., Journal of NeuroImmune Pharmacology, 2, 222-231, 2007 J. P. Michaud et al., Proceedings of the National Academy of Sciences of the United States of America, 110, 1941-1946, 2013 C. Kohchi et al., Journal of Bioscience and Bioengineering, 102, 485-496, 2006 M. Fukasaka et al., PLOS ONE, 10, e0126849, 2015 Y. Kobayashi et al., Anticancer Research, 36, 3693-3698, 2016 J. E. Morley et al., Biochimica et Biophysica Acta, 1822, 650-656, 2012 J. Mehla et al., Journal of Alzheimer's Disease, 39, 145-162, 2014 K. Nakata et al., Nutrition Research and Practice, 5, 435-442, 2011

The inhibitory effect of Aβ deposition in the brain or the improvement of learning function is considered to be useful for the prevention of dementia, especially Alzheimer's disease. The present invention has been made in view of the above-described background, and has a brain function improving agent having an inhibitory effect of Aβ intracranial deposition by lipopolysaccharide, which can be obtained relatively easily by the fermentation culture method (Patent Document 2), or an improvement effect of learning function. To provide food, medicine and medicine.

The present inventors earnestly examined in order to solve the said subject. Pantoea LPS was orally administered to Alzheimer's disease model mice and its effects were investigated. As a result, it was found that the oral administration of Pantoea bacillus LPS significantly reduced the amount of Aβ peptides accumulated in the brain and improved learning function, thus completing the present invention. The brain function improving agent of the present invention is directed to Alzheimer's disease, characterized by containing Pantoea LPS as an active ingredient.

That is, this invention has the following structures.

(1) Brain function improving agent, food or pharmaceutical product which uses lipopolysaccharide which consists of Pantoea bacteria LPS as an active ingredient.

(2) The brain function improving agent, food or medicine according to claim 1, wherein the brain function to be improved is a cognitive function deteriorated due to, for example, or a brain disease according to, for example.

(3) The brain function improving agent, food or medicine according to claim 1, which contains an effective amount of Pantoea bacteria LPS.

(4) The brain function improving agent, food or medicine according to claim 1 for preventing Alzheimer's disease.

(5) The brain function improving agent, food or medicine according to claim 1, which contains Pantoea LPS at a ratio of 0.1 to 1 mg / kg body weight per unit of daily administration.

ADVANTAGE OF THE INVENTION According to this invention, the composition of drugs, foods, etc. which have the preventive effect of Alzheimer's disease, especially the inhibitory effect of A (beta) brain deposition and the improvement of learning function can be provided by pantoea bacterium LPS. Pantobacteria LPS has been confirmed safety in oral or transdermal administration according to the form of food, cosmetics, feed, etc., can be expected to have a small preventive effect of the risk of side effects.

1 shows the results of a training test in an underwater maze test. With five consecutive days of training, the time required to reach the platform is a graph showing shortening in all groups. In addition, the difference between groups was not confirmed.
2 shows a representative swimming trajectory in the probe test in the underwater maze test. The dotted circle indicates the position where the platform was installed.
Fig. 3 shows the results of the time when the mouse stayed in the quadrant region where the platform was installed in the probe test of the underwater maze test. In the PC group fed high fat feed, the reduction of the residence time was confirmed, whereas ingestion of Pantoea bacteria LPS is a result of prolonging the residence time.
4 shows the accumulation of Aβ (Aβ1-40 and Aβ1-42) in the brains extracted from mice after 18 weeks of breeding. In the PC group fed high fat diet, significant increase of Aβ 1-40 was observed compared to NC group, and Pantoea LPS intake was significantly higher in Aβ 1-40 and Aβ 1-42 compared with PC group. This is a result showing that the lowering.

(Form to carry out invention)

The brain function improving agent and the food composition of the present invention contain pantoea LPS.

Herein, except for the case where the pantoea bacterium LPS is specifically described, Pantoea agglomerans of Gram-negative bacteria that coexist in wheat flour are cultured in wheat flour according to the procedure described in Patent Document 2. The lipopolysaccharide is hydrothermally extracted from the cells and the solid content is removed.

Brain function improving agents and foods of the present invention can ameliorate brain dysfunction, in particular cognitive dysfunction due to, for example, or accompanying brain diseases. As a brain disease accompanying a cognitive dysfunction, Alzheimer's disease etc. can be specified, for example.

The pantoea bacterium LPS of the present invention can be applied to humans and non-human mammals (animals such as pigs, cattle, sheep, horses, dogs, cats), birds (poultry such as chickens, turkeys, ducks) and the like.

The route of administration, the dosage form, and the like of the pharmaceutical composition of the present invention can be appropriately designed according to the purpose, symptoms, age, weight, etc. of the subject. Examples of routes of administration include oral administration, transdermal administration, oral administration, subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular administration and the like. Preferably, oral administration, transdermal administration, oral administration. Examples of the dosage form include powders, granules, solutions, capsules, fine granules, pills, syrups, emulsions, and the like. This pharmaceutical composition can be administered orally and is also effective. These formulations, in addition to Pantoea LPS, include a variety of acceptable additives as pharmaceuticals, such as stabilizers, fillers, emulsifiers, extenders, excipients, binders, humectants, disintegrants, surfactants, suspensions, coatings, colorings, flavorings , Flavoring agents, sweetening agents, preservatives, and antioxidants.

The food composition of this invention can be used according to the commercial method in a food composition, using Pantoea bacillus LPS as it is, or mixing with another foodstuff or food component. In addition, the form is not particularly limited either, and may be any of a state of a foodstuff that is usually used, for example, solid (powder, granular, etc.), paste, liquid or suspension. The food composition of the present invention may be a nutritional functional food, a specific health food, a functional labeling food, a health food, a nutritional supplement, a drink, a soft drink, an alcoholic beverage, a supplement, a feed, a feed additive.

In the case of oral ingestion, the brain function improving agent or the food composition of the present invention is preferably formulated so as to contain Pantoea bacillus LPS in the range of 0.1 to 1 mg / kg body weight per adult, and can be administered by dividing once to several times. have. Preferably it is 0.3-1 mg / kg body weight, More preferably, it is 0.5-1 mg / kg body weight. Such doses may be appropriately adjusted depending on various factors such as the subject's age, weight, health condition, method of administration and combination with other agents (or food ingredients). When ingested for the purpose of prophylaxis over a long period, the amount may be smaller than the above range.

Example

Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited to the following example.

Example 1 Oral Administration Test Method of Pantoea LPS in Alzheimer's Disease Model Mice

The pantoea bacteria LPS was prepared and purified according to the fermentation culture method of lipopolysaccharide (Patent Document 2) developed by Soma et al. (Lipopolysaccharide, Pantoea agglomerans <LPS>, Natural Immunology Research Institute). In the experiment, 12-14-week-old male SAMP8 mice (Senescence accelerated mouse-prone 8 / Ta Slc, Nippon SLC Co., Ltd.) were used as Alzheimer's disease model mice, and were divided into four groups according to body weight after one week of preliminary breeding. A feed containing 35% fat (research diet) was given and water containing 0.3 or 1 mg / kg body weight / day of Pantoea LPS was given by oral administration by free negative water. In addition, the control group was given a feed containing 35% fat or a feed containing 4% fat (research diet) and watered in the same manner. The breeding of mice was carried out in an animal facility in a temperature-humidity controlled environment under free feeding, free negative water, 12 hours light irradiation / 12 hours dark environment. After breeding for 17 weeks, the underwater maze test shown below was performed for 1 week, and the learning function was evaluated. The day after the end of the underwater maze test, blood was drawn from the heart. White fat cells around the brain, liver, and testis were extracted. This animal experiment was approved by the Kagawa University Animal Experiment Committee.

In this example, male SAMP8 mice were used as Alzheimer's disease model mice. This mouse was used as an aging promoting model mouse because it exhibits Alzheimer's disease-like symptoms such as an increase in the amount of Aβ deposition in the brain and a decrease in learning function, for example (Non-Patent Document 9). In addition, high-fat ingestion has been reported to accelerate the development of Alzheimer's disease by inducing symptoms of type 2 diabetes mellitus, for example, an increase in blood glucose value and an increase in HbA1c value when fasting (Non-Patent Document 10).

In this example, mice were divided into the following four groups.

(1) NC group: Free feed containing 4% fat (low fat diet) and water.

(2) PC group: Free intake of water containing 35% fat (high fat feed) and water.

(3) Pantoea LPS 0.3 mg / kg body weight / day group: A high-fat diet and water containing Pantoea LPS (0.3 mg / kg body weight / day) were allowed to be ingested freely.

(4) Pantoea LPS 1 mg / kg body weight / day group: A high-fat diet and water containing Pantoea LPS (1 mg / kg body weight / day) were allowed to be ingested freely.

[Underwater maze test]

(1) device

Water (23 ± 1 ° C.) was placed in a cylindrical pool (100 cm in diameter and 40 cm in depth) and the transparent platform (10 cm in diameter) was settled down to 1 cm below the water surface. Commercial black ink was added to the water of the pool so that the swimming mouse could not see the platform. A commercial digital camera was installed just above the pool surface, and the swimming of the mouse was recorded as a moving picture. The swimming trajectory was analyzed according to the method described in "Neuroinformatics, 14, 479-481, 2016" using image analysis software Aminal Tracker.

(2) procedure

The day before the test, the mice were swimming once each to familiarize themselves with the pool. The procedure allowed the mouse to rest on the platform fixed at 1cm above the surface for 20 seconds, and then freely swim for 30 seconds. The mouse was then guided onto the platform with the hands of the experimenter and left to stand for 20 seconds. In addition, when entering the pool, the mouse was obtained in the direction of the wall of the pool, and the experimenter quickly moved from the mouse to an invisible position. On days 1-5, the mouse was trained to memorize the position of the platform. Training was performed four times a day in succession. The procedure for training was to move the mouse into the pool at any position, swim for 60 seconds, and search for a platform installed 1 cm below the surface of the water. The time required to reach the platform was recorded and 60 seconds if it could not be reached. In addition, mice that did not reach the platform in time were guided to the platform by the experimenter's hand. After reaching the platform, it was left standing for 20 seconds and the mouse was taken out of the pool. The probe test was done on the 6th day. The probe test removed the platform from the pool, allowed the mice to swim for 60 seconds, and measured the residence time within each quadrant region of the pool. In addition, the probe test was performed once for each mouse.

Blood glucose values of blood were measured using a blood glucose value measuring device (Roche diagnostics) and HbA1c using an enzyme method measurement kit (Sekisui Medical). Triglycerides, total cholesterol, LDL cholesterol, and HDL cholesterol in plasma were measured using an enzyme method measurement kit (Wakkoyaku Kogyo). Oxidative LDL in plasma was measured using an ELISA kit (Kamiya Biomedical Company). Aβ peptide accumulation in the brain was measured using an ELISA kit (Wako Pure Chemical Industries, Ltd.). According to the protocol of the instruction manual, Aβ fractions were prepared from the extracted mouse cerebral samples, and Aβ peptides (Aβ1-40) each consisting of Aβ1-42 and 40 residues were quantified. The results are expressed as mean and standard error of the mean (SEM). One-way batch variance analysis was also performed according to Tukey-Kramer's multiple comparisons. Different symbols indicate a significant difference at P <0.05.

[result]

After 18 weeks of breeding, body weight was significantly different between NC and PC groups, but not with Pantoea LPS-administered group. Fasting blood glucose levels were significantly increased in the PC group compared to the NC group, and significantly lower in the Pantoea LPS-administered group compared to the PC group. Similarly, HbA1c was significantly decreased in the PC group compared to the NC group, and significantly lower in the Pantoea LPS 1 mg / kg body weight / day group compared to the PC group. In addition, the hepatic weight and the testicular upper body white fat weight were clearly increased in the PC group compared to the NC group, and significant decrease in the pantoea LPS-administered group compared to the PC group (Table 1). From this, mice ingested high-fat diet develop symptoms of type 2 diabetes-like symptoms (such as an increase in blood glucose value and HbA1c value), and it can be seen that glucose metabolism function is improved by administration of pantoea bacteria LPS. have.

As a result of analyzing blood lipids, a clear decrease in total cholesterol and LDL cholesterol was confirmed in the pantoea bacteria LPS-administered group. HDL cholesterol was clearly elevated in the pantoea bacteria LPS1 mg / kg body weight / day group compared with the PC group. In addition, the oxidized LDL of plasma was lower than the Pantoea LPS 1.0mg / kg body weight / day compared to the PC group than the Pantoea LPS 0.3mg / kg body weight / day (Table 1). From this, it can be seen that in SAMP8 mice fed high fat diet, oral administration of pantobacterial LPS improves glucose metabolism and lipid metabolism. So far, Nakada et al. Have been orally administering pantoea LPS combination (0.02% w / w) tea beverages to diabetic model mice (KK-Ay), compared to the control group that was given water, and fasting blood sugar when compared to the control group. Significantly lowered values, and also in clinical trials (double-blind randomized comparative trials) of the pantoea bacteria LPS blended tea beverages, reported a decrease in LDL cholesterol and an increase in HDL cholesterol by ingestion of the beverage. (Non-Patent Document 10).

As a result of the training test of the underwater maze test, a shortening of the time required to reach the platform was confirmed in any group by the training for 5 days, but no difference was found between the groups (FIG. 1). The probe test was done the day after the training test. The swimming trajectory of the mouse is shown in Fig. 2 representative data for each group. In the residence time of the quadrant area in which the platform was installed, a clear decrease was confirmed in the PC group compared with the NC group. In addition, Pantoea LPS 1.0mg / kg body weight / day than Pantoea LPS 0.3mg / kg body weight / day was longer compared to the PC group (Fig. 3). From the result of learning function evaluation based on the underwater maze test, it turns out that the brain function regarding the learning function of Alzheimer's disease onset (aging promotion) model mouse improves by oral administration of Pantoea bacillus LPS.

As a result of measuring Aβ accumulation in the brain by ELISA, it was confirmed that Aβ1-40 was significantly increased in the PC group compared to the NC group, and significantly lowered in the pantoea LPS-administered group compared to the PC group. Moreover, about Aβ1-42, although the significant difference was not confirmed between NC group and PC group, the significant fall was confirmed by the pantoea LPS administration group compared with PC group (FIG. 4). From this, it can be seen that Pantoea bacterium LPS suppresses the onset of Alzheimer's disease (decreases in learning function, etc.) by inhibiting accumulation of Aβ in the brain.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

In addition, the indication of the Japan patent application 2017-105133 for which it applied on May 28, 2017 including specification, a claim, and drawing is taken in as a reference as it is here.

Claims (5)

Brain function improving agent, food or pharmaceutical product using lipopolysaccharide consisting of Pantoea bacterium LPS as an active ingredient. The method of claim 1,
Food or pharmaceuticals, characterized in that the brain function to be improved is a cognitive function that is degraded due to, or according to, for example, a brain disease. The method of claim 1,
A brain function improving agent, food or medicine comprising an effective amount of Pantoea LPS. The method of claim 1,
Brain function improving agent, food or medicine, characterized in that for preventing Alzheimer's disease. The method of claim 1,
A brain function improving agent, food or medicine comprising Pantoea LPS at a ratio of 0.1 to 1 mg / kg body weight per day dosage unit.

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